Methods of treating viral infections using berry juice fractions

ABSTRACT

The present invention concerns the use as anti-viral agents of isolated fractions from berry juice of the plant genus  Vaccinium . Two specific fractions are provided: (a) polymeric material having a molecular weight≧12,000; and (b) proanthocyanidins-containing fraction. 
     Thus, the invention provides methods of treating a subject against a viral infection; the method comprises providing said subject with at least one of the above isolated fractions in an amount effective to prevent production of a viral infection in said subject. 
     A specific and preferred viral infection according to the invention is an influenza virus infection.

FIELD OF THE INVENTION

This invention relates to fractions isolated from Vaccinium plantsberries having an anti-viral activity. Specifically, to isolatedfractions from cranberry juice which have anti-viral characteristics.

PRIOR ART

The following is a list of prior art which is considered to be pertinentfor the state of the art in the field of the invention.

-   Ofek, I., et al. New Eng. J. Med. 324:1599 (1991);-   Ahuja, S., et al., J. Urol. 159:559-562 1998;-   Burger, O., et al. FEMS Immunol. Med. Microbiol. 29:295-301 (2000);-   Foo, L. Y., et al. Phytochemistry 54:173-181 (2000(a));-   Weiss, E. I., et al. JADA 129:1719-1723 (1998);-   Zafriri, D., et al. AntiMicrob. Agents Chemothe. 33:92-98 (1998);-   Weiss E. I., et al. FEMS Microbiol Lett. 232:89-92 (2004);-   Foo, L. Y. et al. Escherichia coli J. Nat. Prod., 63:1225-1228    (2000(b))-   U.S. Pat. No. 5,840,322;-   U.S. Pat. No. 6,303,125;-   U.S. Pat. No. 6,843,993;-   U.S. Pat. No. 5,474,774.

BACKGROUND OF THE INVENTION

Myxoviridae

Influenza viruses belong to a broad family of RNA viruses, theOrthomyxoviridae (myxo=mucus) viruses. Orthomyxoviridae viruses as wellas the closely related family or RNA viruses, Paramyxoviridae viruses,are characterized by a negative-stranded RNA genome (segmented ornon-segmented, respectively), having, an inner ribonucleoprotein (RNP)core surrounded by a lipid bilayer membrane from which spikes protrude.The spikes are of three kinds: a hemagglutinin (HA) which agglutinateserythrocytes, an enzyme neuraminidase (NA) which releases the virus fromcells and a small number of copies of the M2 protein that serves as anion channel. These spikes in influenza (in Parmyxoviridae virus theseare HN and F) are involved in hemagglutination, hemolysis oferythrocytes etc. and cleavage of the receptor (on the cell)anti-receptor (on the virus) bond, and reflect the ability of the virusto enter the nucleoprotein core into cells.

Influenza is a highly communicable acute respiratory disease thatpredisposes to a number of complications, resulting in a severeworld-wide economic burden. Prevention and control of both the annualinfluenza epidemics and its infrequent but severe pandemic outbreaks arehitherto achieved by the use of vaccines and newly emerging antiviraldrugs.

Unfortunately, the vaccines provide sometimes lower than desirableprotection, particularly in the immuno-compromised and the elderly, thetwo most susceptible subpopulations (Keren. G., et al. J. Med. Virol.25:85-89 (1988); Admon, D., et al. Vaccine 15:1518-1522 (1997)).Furthermore, the vaccines currently available are designated forintramuscular injection, resulting mainly in serum antibodies.

As to antiviral drugs, two classes of antiviral drugs are used:

(i) Anti-M2 inhibitors amantadine and rimantadine, effective against Astrains only (World Health Organization, WHO Org. Rep. Ser., Geneva.642:1-63 (1980)). A reduction in the severity and duration of the signsand symptoms is recorded when they are administered within 48 hrs ofdisease onset (Centers for Disease Control and Prevention, 1996.Prevention and control of influenza: Recommendation of the advisorycommittee on immunization practices (ACIP). MMWR45(RR-5), 1-24);

(ii) Neuraminadase inhibitors, effective against both A and B viruses.As prophylactics, these inhibitors are 70-90% effective and may shortenthe duration of illness by 1.5 days when used within the first 48 hr(Treanor, J., Falsey, A., Antiviral Res. 44:79-102 (1999)).

Vaccinium Genus

Vaccinium is a genus of evergreen or deciduous berry-bearing ericaceousshrubs including the various kinds of blueberries and the truecranberries.

Physicians have long recommended consumption of cranberry juice to avoidurinary tract infections. It was hypothesized that the prevention ofbacterial infections is due to the inhibition of E. coli adhesion touroepithelial cells by cranberry constituents (Ofek et al., 1991,ibid.). Studies have shown that cranberries contain high and lowmolecular weight constituents [nondialyzable material (NDM) andproanthocyanidins, respectively], which act in vitro to inhibit theadhesion of diverse microbial species (Ahuja et al., 1998 ibid.; Burgeret al., 2000, ibid.; Foo et al., 2000a, ibid.; Weiss et al., 1998 ibid.;Zafriri et al., 1998, ibid). It has been suggested that cranberryproanthocyanidins are one of the active anti-adhesion agents (Foo etal., 2000a, ibid.).

SUMMARY OF THE INVENTION

The present invention is based on the finding that fractions, isolatedfrom cranberry juice, exhibit anti-viral activity. This anti-viralactivity was demonstrated in vitro, by preventing virus adsorption tored blood cells thus inhibiting hemagglutination (HA) and to cells incultures interfering in virus propagation in host cells, as well as invivo, by exhibiting an increased survival rate of infected mice. Thus,it was envisioned that fractions from juice from berries of the plantgenus Vaccinium may be have a beneficial effect against viralinfections.

Thus, according to a first aspect, the invention provides a method oftreating a subject against a viral infection, the method comprisesproviding said subject with an amount of a polymeric material having amolecular weight ≧12,000-14,000 and isolated from juice from berries ofthe plant genus Vaccinium, the amount of said polymeric material beingeffective to prevent production of a viral infection in said subject.

According to second aspect, the invention provides a method of treatinga subject against a viral infection comprising providing said subjectwith an amount of a proanthocyanidins-containing fraction isolated fromjuice from berries of the plant genus Vaccinium, the amount of saidproanthocyanidins-containing fraction being effective to preventproduction of viral infection in said subject.

A specific and preferred viral infection according to the invention isan influenza virus infection.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to understand the invention and to see how it may be carriedout in practice, a preferred embodiment will now be described, by way ofnon-limiting example only, with reference to the accompanying drawings,in which:

FIG. 1 is a graph showing the dose dependent effect of NDM on inhibitionof virus HA activity.

FIG. 2 is an image showing the prevention of virus-induced cytopathiceffect by NDM, and in which MDCK monolayers were inoculated withB/Yamanashi virus suspension (rows A, B and C) only or with a mixture ofNDM (250 μg/ml) and B/Yamanashi/166/98 virus, (rows D, E and F). Bluestaining (observed as darker wells) indicates that the cells are intact;staining is not obtained if the cells are destroyed.

FIG. 3 is a bar graph showing the levels of INFγ in the serum of micefollowing influenza challenge and NDM treatment.

DETAILED DESCRIPTION OF THE INVENTION

According to a first aspect, the invention provides a method of treatinga subject against a viral infection; the method comprises providing thesubject with an amount of a polymeric material having a molecularweight≧12,000-14,000 kDa, preferably≧14,000 kDa and isolated from juicefrom berries of the plant genus Vaccinium, the amount of said polymericmaterial being effective to prevent production of a viral infection insaid subject. Due to the relatively high molecular weight of thesubstances forming part of this isolated polymeric material, this aspectof the invention is referred to herein by the term “non-dialyzablematerial aspect”, or “NDM aspect” of the invention.

According to a second aspect, the invention provides a method oftreating a subject against a viral infection; the method comprisesproviding said subject with an amount of a proanthocyanidins-containingfraction isolated from juice from berries of the plant genus Vaccinium,the amount of said proanthocyanidins-containing fraction being effectiveto prevent production of viral infection in said subject. Due to theproanthocyanidins content in this isolated fraction, this aspect of theinvention is referred to herein by the term “proanthocyanidins aspect”or “PAC aspect” of the invention.

Notwithstanding the above, when appropriate, the isolated polymericmaterial, according to the NDM aspect of the invention, and theproanthocyanidins-containing fraction, according to the PAC aspect ofthe invention, are collectively referred to by the term “isolatedfraction”.

The isolated fraction according to the invention comprises at least oneof the following characteristics:

-   -   it is capable of inhibiting viral induced hemagglutination;    -   it is capable of inhibiting viral adsorption to a susceptible        host cell;    -   it is capable of inhibiting viral penetration into a susceptible        host cell (e.g. by endocytosis);    -   it is capable of inhibiting viral replication, thereby        preventing propagation of viral progenies.

The isolated fraction is effective for treatment of a subject against aviral infection. According to the present invention the term “treatment”denotes any physiological effect resulting in the therapeutic treatmentof a viral infection or prevention (prophylactic treatment) of theformation of a viral infection in a subject exposed to an infectiousvirus. Thus, in the context of the present invention, treatment refersto at least one of the following: inhibition of viral adsorption tosusceptible cells, inhibition of viral penetration into susceptiblecells, inhibition of viral replication, all of which result in adecrease in viral load (thereby less antibody response) in an infectedsubject, preferably, total elimination of the virus from the subject.Treatment preferably results in the improved and more rapid recovery ofthe subject from the viral infection or the prevention of the infectionfrom occurring.

The effective amount of each of the isolated fraction, i.e. thepolymeric material as well as the proanthocyanidins-containing fraction,is determined by such considerations as may be known in the art. Theamount must be effective to achieve the desired therapeutic orprophylactic effect described above. The amount is typically determinedin appropriately designed studies and the person versed in the art willknow how to properly conduct such studies in order to determine therequired effective amount.

With respect to the NDM aspect of the invention a typical concentrationrange of the polymeric material in a single dose administered to asubject in need is between 1 μg/ml and 5 mg/ml within a suitablephysiologically acceptable carrier, with a preferred concentrationbetween 10 μg/ml and 500 μg/ml. With respect to the PAC aspect of theinvention a typical concentration range of theproanthocyanidins-containing fraction in a single dose administered to asubject in need is between 5 μg/ml and 500 μg/ml within a suitablephysiologically acceptable carrier.

The isolated fraction according to the invention may be obtained fromany member of the plant genus Vaccinium. As well appreciated by thoseversed in the art of botanic, the Vaccinium consists of about 450species of evergreen and deciduous shrubs. Some kinds are grown for thebeauty of their fall leaves and some for their attractive flowers anddeliciously edible fruits. Many different names have been given to thenumerous varieties that produce edible fruits which may be used inaccordance with the present invention. Some non-limiting examplesinclude the Blueberry, Bilberry, Cowberry, Cranberry, Crowberry;Farkleberry, Lingonberry, Partridgeberry, Huckleberry Whortleberry, andSparkleberry.

The invention makes use of isolated fraction which may be obtained fromberries juice as well as from berries juice concentrates. Berries juiceand berries juice concentrates may be prepared by methods known in theart or obtained from commercially available products. A specific andpreferred juice is cranberry juice which is commercially available.

According to the NDM aspect of the invention, the isolated polymericmaterial may be obtained and is characterized as described by Ofek, I.,et al. (Ofek et al. et al. Adv. Exp. Med. Biol. 408:179-183 (1996); andU.S. Pat. Nos. 6,303,125, 5,840,322 and 6,843,993, all of which areincorporated herein by reference in their entirety). Accordingly, theisolated polymeric material is further characterized by at least one ofthe following parameters:

-   -   it has an elemental analysis of carbon 43-51%, hydrogen 4-5%, no        nitrogen, no sulfur and no chlorine, preferably 56.6% carbon and        4.14% hydrogen;    -   it has a characterizing NMR fingerprint spectrum as set forth in        the aforementioned U.S. Pat. Nos. 6,303,125, 5,840,322 and        6,843,993, all being incorporated herein by reference in their        entirety;    -   it has an ultraviolet spectrum with an absorption peak at 280 nm        in neutral or acidic pH solution which is absent in alkali        solutions.    -   it exhibits tannin-like properties;    -   it is highly soluble in water;    -   it is devoid of proteins, carbohydrates and fatty acids;    -   it has less than 0.02 mg/ml of phenolic residues at a        concentration of 0.1 mg/ml.

As should be appreciated, there are many alternative ways to produce apolymeric material, having one or more of the aforementioned physicaland chemical characteristics. According to one embodiment of theinvention, the polymeric material is obtained by subjecting cranberryjuice or cranberry juice concentrate (as an exemplar species ofVaccinium) to dialysis at against double distilled water (ddWater) usingdialysis tubing with a≧12,000 kDa-14,000 kDa molecular cut-off andcollecting the non-dialyzable material (NDM) remaining in the dialysistubing. This remaining NDM may then be lyophilized to obtain a drypowder which may be fractionated on a resin, e.g. polyacrylamide resincolumn. The active polymeric material may then be eluted from the columnwith water and once again lyophilized to obtain a dry polymericmaterial.

The non-dialyzable material, NDM, was assayed for polyphenol compoundsas follows:

Polyphenol Assay: A modified Folin-Denis test [Folin and Denis, 1912]was used to detect all hydroxylated phenolic compounds [Mehansho et al,1987]. As a control standard commercially available tannin andanthocyanin was obtained from Sigma Chemical Co. (St. Louis, Mo.). Thetest was sensitive for phenolic compounds concentrations down to 0.02mg/ml tanin in standard

Results: NDM at 0.1 mg/ml concentration (repeated twice) showed lessthan 0.02 mg/ml of phenolic residues. Therefore, NDM does not containphenolyic residues found typically in tannins and anthocyanins by thistest.

Referring to the PAC aspect of the invention, theproanthocyanidins-containing fraction is further characterized by one ormore of the following characteristics:

-   -   it is enriched with polyphenol and flavonoid compounds;    -   it has a characteristic elution peaks at 230 nm, 280 nm, and 360        nm, as described by Foo et al. [Foo L. Y., et al. 2000(a);        Foo L. Y., et al. 2000(b)], incorporated herein by reference in        its entirety;    -   it has a benzoic acid content of less than about 0.01 per gram        dry powder as described by Foo et al. [Foo L. Y., et al.        2000(a); Foo L. Y., et al. 2000(b)], incorporated herein by        reference in its entirety.

The proanthocyanidins-containing fraction may be obtained as describedby Foo et al. [Foo L. Y., et al. 2000(a); Foo L. Y., et al. 2000(b)],incorporated herein by reference in its entirety. Further, theproanthocyanidins-containing fraction is commercially available, and maybe obtained, for example, from Ocean Spray [Ocean Spray Cranberries,Inc, Lakeville-Middleboro, Mass. 02349].

The viral infection according to the invention is an infectionpreferably localized within the respiratory tract, either the upperrespiratory tract, lower respiratory tract, or both. Non-limitingexamples of viruses causing an infectious disease within the respiratorytract include members of the Orthomyxoviridae family or Paramyxoviridaefamily of viruses.

According to one embodiment, the virus causing the infection is a memberof the Orthomyxoviridae family. According to a specific embodiment, thevirus is selected from Influenza viruses.

According to another embodiment, the virus is a member of theParamyxoviridae family. According to one embodiment, the virus isselected from Para-Influenza Types 1, 2, 3 and 4 Virus and RespiratorySyncytial Virus (RSV). Other members of the group include Humanmetapneumovirus and Newcastle Disease Virus (NDV) which infect poultry.

According to yet another embodiment of the invention, the virus ischaracterized by infecting cells carrying on their surface receptorscomprising sialyl sugar chains, the sialyl sugar chains bearingreceptors being involved in the adsorption of the virus onto thesusceptible cell thereby causing infection. Sialyl sugar-chains bearingreceptors are found, inter alia, on the surface of influenza virus aswell as on the surface of other viruses, all forming part of the presentinvention.

It is commonly known that viral infections may be transmitted fromanimals to humans. Thus, the invention is also applicable for treatingsubjects where the virus is an animal-type virus being transmitted froman animal to humans. For example, it is known that influenza A virus maybe found in many animals, such as ducks, chickens, pigs, whales, horses,and seals which may be transmitted to humans resulting in an infectiousdisease.

Further, the invention is applicable for treating non-human animals fora viral infection. A non-human animal viral infection of particularinterest includes the Avian Influenza viruses (also known by the term“Bird Flu”). Avian influenza virus can be transfer to human with highmorbidity rate and therefore a therapeutic treatment thereagainst is ofhigh importance.

The isolated fraction may be administered in various ways suitable foranti-viral therapy. It should be noted that it can be administered asthe extracted fraction per se (e.g. as a powder), or within a suitablecarrier suitable for a selected delivery route. While the isolatedfraction may be administered by any suitable route, e.g. orally, apreferred mode of administration of the isolated fraction according tothe invention is by intranasal application, preferably, by inhalation.

According to one embodiment, the isolated fraction is administered inthe form of a solution, e.g., water or isotonic saline, buffered orunbuffered, or as a suspension, for intranasal administration as dropsor as a spray. Preferably, such solutions or suspensions are isotonicrelative to nasal secretions and of about the same pH, ranging e.g.,from about pH 4.0 to about pH 7.4 or, from pH 6.0 to pH 7.0. Buffersshould be physiologically compatible and include, simply by way ofexample, phosphate buffers. For example, a representative nasaldecongestant is described as being buffered to a pH of about 6.2(Remington's Pharmaceutical Sciences, Ed. By Arthur Osol, p. 1445(1980)). Of course, the ordinary artisan can readily determine asuitable saline content and pH for an innocuous aqueous carrier fornasal administration.

Other, non-limiting examples of intranasal dosage forms containing theisolated fraction include nasal gels, creams, pastes or ointments with aviscosity of, e.g., from about 10 to about 3000 cps, or from about 2500to 6500 cps, or greater, which may provide a more sustained contact withthe nasal mucosal surfaces. Such carrier viscous formulations may bebased upon, simply by way of example, polymeric carriers such asalkylcelluloses and/or other biocompatible carriers of high viscositywell known to the art (see e.g., Remington's, cited supra). The carriercontaining the isolated fraction may also be soaked into a fabricmaterial, such as gauze, that can be applied to the nasal mucosalsurfaces to allow for active substances in the isolated fraction topenetrate to the mucosa.

Other ingredients, such as art known preservatives, colorants,lubricating or viscous mineral or vegetable oils, perfumes, natural orsynthetic plant extracts such as aromatic oils, and humectants andviscosity enhancers such as, e.g., glycerol, can also be included toprovide additional viscosity, moisture retention and a pleasant textureand odor for the formulation.

Further, for nasal administration of solutions or suspensions of theisolated fraction, various devices are available in the art for thegeneration of drops, droplets and sprays. For example, solutionscomprising the isolated fraction can be administered into the nasalpassages by means of a simple dropper (or pipet) that includes a glass,plastic or metal dispensing tube from which the contents are expelleddrop by drop by means of air pressure provided by a manually poweredpump, e.g., a flexible rubber bulb, attached to one end. Fine dropletsand sprays can be provided by a manual or electrically poweredintranasal pump dispenser or squeeze bottle as well known to the art,e.g., that is designed to blow a mixture of air and fine droplets intothe nasal passages.

The following examples, which are in no way intended to limit the scopeof the present invention, illustrate the preparation of cranberryisolated fractions and their effect against influenza virus infection.Obviously, many modifications and variations of the present inventionare possible in light of the above teaching. It is therefore, to beunderstood that within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described hereinbelow.

SPECIFIC EXAMPLES

In the following examples, the activity of NDM on influenzavirus-mediated red blood cells (RBC) hemagglutination (HA), in vitroreplication of the virus and in vivo infectivity of the virus, weretested.

Cranberry Juice

Cranberry juice from the American cranberry, Vaccinium macrocarpon, anda proanthocyanidin-rich fraction were obtained from Ocean Spray, Inc.[Ocean Spray Cranberries, Inc, Lakeville-Middleboro, Mass. 02349]

To obtain the polymeric material having molecular weight≧12,000 kDa, thejuice was dialyzed at 4° C. for 10 days against distilled water, changedten times, in dialysis bags of≧12,000-14,000 MW cut-off and lyophilized.The non-dialyzable material (NDM) exhibits tannin-like properties, ishighly soluble in water, devoid of proteins, carbohydrates and fattyacids and contains 56.6% carbon and 4.14% hydrogen (Ofek et al., 1996,ibid).

Influenza Virus

The following influenza virus strains were used:

1. A/PR/8/34 H₁N₁ grown in the allantoic sac of 10-11-day-old cells eggsto a 1:1024-2048 HA titer, or grown in Madine-Darby canine kidney (MDCK)cells to a 1:128 HA titer.

2. A/H₁N₁ (1:128 HA titer) and A/H₃N₂ (1:256 HA titer), clinicalisolates grown in MDCK cells.

3. A/Panama 2007/99 H₃N₂ adapted to MDCK cells (1:256HA titer)

4. B/Yamanashi/166/98, grown in MDCK cells (1:256-512 HA titer). Toevaluate replication inhibition, viruses cultivated in MDCK cells wereused.

To determine HA, 0.1 ml of twofold dilutions of each virus suspension inphosphate-buffered-saline (PBS) was mixed with 0.1 ml of a 0.5% chickenRBC or 1% sheep RBC or human 1% O RBC suspension and scored after 30 minincubation at room temperature (Sever, J. L., J. Immunol. 88:320-329(1962)).

HA Determination

The HA data are summarized in the following Table 1:

TABLE 1 Viral Hemmagglutination and replication in NDCK cells HAU ^(a)MDCK cells (log 10) ^(b) Viral stain Untreated Treated Untreated TreatedA/PR8/34 (i) 16 <1 NP NP egg (ii) 16 2-4 6.5 1.5 MDCK A/H₃N2 16 4-8 6.51.5 A/H1N1 16 4-8 6.0 1.5 B/Yamanashi/ 16 <1 7.5 1.5 166/98 A/H5N3(Avian) 16 2-4 6.5 2.5 ^(a) Hemagglutination units of treated virus(preincubated with 125 μg/ml of NDM), compared with that of untreatedvirus. ^(b) TCID₅₀ (Tissue Culture Infecting Dose) in MDCK cells oftreated virus (preincubated with 250 μg/ml of NDM), compared with thatof untreated virus. NP = not performed

Preincubation of NDM (125 μg/ml) with A/PR/8 (egg-grown) or B/Yamanashi(grown in MDCK cells) strains inhibited virus-induced HA. This wasreduced from 16 HA units (HAU) to <1 in NDM-containing virus suspensions(100%). NDM at 125 μg/ml reduced the 16 HAU of the two clinical isolatesA/H₁N₁ and A/H₃N₂ to 4-8 units. A/PR/8 grown in MDCK cells was lesssensitive to 125 μg/ml NDM; the 16HAU were reduced to 2-4 HAU (Table 1).

A higher NDM concentration (400 μg/ml) was needed to reduce the HAU ofthe MDCK cell-grown A/PR/8 strain from 16 to <1 units (not shown). Chessboard titration of virus densities and decreasing NDM concentrationsrevealed that as little as 4, 16, 64 and 128 μg/ml NDM were needed tocompletely inhibit HA induced by 8, 16, 32, 64 respectively, HAU ofvirus A/PR8/34. The results indicate a highly significant r²=0.980correlation between HAU and NDM concentration required to completelyinhibit HA (FIG. 1). HA inhibition was also observed using sheep orhuman RBC, consistent with the notion that the target for NDM is thevirus.

Virus Replication

To test inhibition of virus replication, MDCK cells were grown in DMEMmedium supplemented with 10% inactivated FCS and antibiotics (100μg/mlpenicillin G and 100 μg/ml streptomycin). For the assay, cells weregrown in 12-well culture plates (Nunc, Roskilde, Denmark) in ahumidified atmosphere with 5% CO₂ at 37° C., and used when confluentmonolayers had formed (48 hrs). NDM inhibition of virus infectivity invitro was tested by incubating 250 μg/ml NDM with virus for 2 h at roomtemperature. Tenfold serial dilutions in medium without serum wereinoculated in triplicate wells containing MDCK monolayers. The virus wasallowed to adsorb for 1 hr at 37° C. The inoculum was removed and mediumsupplemented with 1% serum and trypsin 2 μg/ml (×2 crystallized) wasadded. Following 4 days incubation at 37° C. in a 5% CO₂ incubator, themonolayers were examined for cytopathic effect (CPE) (FIG. 2) and therespective supernatants were assayed for HA. HA was evident at adilution of up to 10⁻⁷ in the control wells, whereas in wells infectedwith the virus-NDM suspension, hemagglutination was observed only at10⁻¹. For the CPE, the monolayers were washed with PBS to remove deadcells and debris, fixed with cold methanol and stained with crystalviolet to determine the integrity of the monolayer. The CPE was observedin all the control wells (FIG. 2 rows A, B and C) at a 10⁻⁷ viraldilution whereas in the virus-NDM suspension the CPE was observed at10⁻¹ (FIG. 2 rows D, E and F). The observed CPE correlated with the HAresults (not shown), allowing calculation of tissue culture infectivedose (TCID₅₀, Reed, L. J., Muench, H. A., Am. J. Hyg. 27:493-497(1938)). NDM reduced the B/Yamanashi titer from 10^(7.5) to 10^(1.5)(Table 1). Significant inhibition of infectivity by NDM was demonstratedfor all the virus strains tested (p<0.001, Fisher's Exact Test): theA/H₃N₂ was reduced from 10^(6.5) to 10^(1.5), A/H₁N₁ from 10^(6.0) to10^(1.5) and A/PR8 (grown in MDCK cells) from 10^(6.5) to 10^(1.5)(Table 1). When A/H₃N₂ and NDM were added simultaneously, the TCID₅₀titer dropped from 10^(6.5) to 10^(2.0) and from 10^(6.0) to 10^(1.5)when A/PR8 was tested. These data suggest that the NDM-virus interactionwas virtually instantaneous and that pre-incubation is not required.Inhibition of infectivity in MDCK cells was similar for the A strains(4.5-5 logs), B/Yamanashi appeared to be more sensitive (6 logs).

Viral Infectivity

To evaluate the potential of the NDM in the therapy of viral infection,the cells were first exposed to viral suspensions to allow adsorptionfollowed by penetration into the cells for 1 hr. NDM (100 μg/ml) wasadded at various post infection time intervals as indicated in Table 2.Viral TCID₅₀ was determined in the treated cultures following 4 days and6 days incubation by assaying the supernatants for HA activity.

The results in Table 2 show that NDM reduced virus TCID₅₀ for the entiretime of the follow up (6 days post infection). This effect was mostaccentuated when the NDM was added several times to the infected MDCKmonolayer (Table 2). These results demonstrate that NDM can reduce orinhibit virus replication indicating inactivation of newly formed virus.

TABLE 2 Effect of NDM post infection Time (h) of NDM treatment postviral adsorption^(s) Viral 1 + 6 + stain Control 1 6 24 24 A/Panama3.5/3.5 <1/3 1.5/2.5 2/2.5^(b) <1/<1^(c) H₃N₂ B/ 4.5/4.5  2.5/3  2/3^(b) 3/3^(b)    <1/1.5^(c)   Yamanashi ^(a)NDM, 100 μg/ml, was addedin each treatment. ^(b)Logistic regression at a confidence interval of95% yielded odds of 42-91. ^(c)Logistic regression at a confidenceinterval of 95% yielded odds of ≧2110.

The effect of cranberry proanthocyanidins-containing fraction wascompared with that of NDM. A four- to fivefold higher proanthocyanidinconcentration was required to completely inhibit 16HA units of A/PR8(grown in egg), providing evidence that that NDM is significantly morepotent (at least six times more active (weight per volume)) thancranberry proanthocyanidins.

Differentiation Between Cranberry Fractions

Comparing virus induced hemagglutination the anti-adsorption activity ofthree cranberry fractions:

1. prothoantocyanidine (PAC)

2. non dialyzable material (NDM) prepared cranberry from juice

3. cranberry powder (CHEP) prepared from press-cake (CHEP is an OceanSpray Inc. product now used by the NIH grantees of the NICCAM projects).

Hemagglutination caused by influenza virus reflects the activity of theviral adsorption which mediates attachment as the first event beforeentry of the virus into the cells. Thus agent(s) which inhibithemagglutination caused by the virus are of potential therapeutic value.

Based on the data shown in Table 3 it can be determined that both thePAC and NDM fractions inhibit viral-induced haemagglutination. CHEP hadno effect on virus induced hemagglutination.

TABLE 3 Effect of cranberry fractions on influenza virus inducedhemagglutination Concentration ^(a) CHEP PAC NDM 500 ± ^(b) + + 250− + + 125 − + + 62.5 − + + 31.2 − ± + 15.6 − ± ± 7.8 − ± ± 3.9 − − ±1.95 − − ± Control + + + ^(a) concentration [μg/ml] in the reactionmixture of the tested fractions. ^(b) + = 100% inhibition ofhemagglutination; ± = partial inhibition; − = no inhibitionIn vivo Anti-Viral ActivityEffect of Intranasal Administration (Inhalation) of CranberryConstituents on Influenza-Virus Infection in Mice.

The virus (A/PR/8/34) was prepared at final concentration of 2×10⁶EID₅₀. NDM was prepared by dissolving the NDM in ddH₂O to a finalconcentration of 500 μg/ml. The total amount of inhaled solution wasadjusted to 80 μl with PBS in the desired concentration. All inhalationswere done under ketamine anesthesia with sterile tips.

Three groups of male Balb/c mice 4-5 weeks old—10 mice each:

-   -   Viral challenge: 10⁶ EID₅₀ in 80 μl of PBS    -   Mixed NDM+Virus: 20 μg NDM mixed with virus in 80 μl of PBS        before administration.    -   Control: NDM 20 μg per animal in 80 μl of PBS

Analyzed clinical parameters during 14 days of the experiment were:

-   -   Blood for cytokines and anti-influenza antibodies    -   Lungs for histology and virus titers

The effect of intranasal administration (by inhalation) of cranberryconstituents on influenza-virus infection in mice is summarized in thefollowing Tables 4 to 7 and in FIG. 3. Specifically, the marked effectof intranasal administration of NDM on the infectious process induced byInfluenza virus as demonstrated by:

-   -   1. Morbidity and mortality (Table 4)    -   2. Histopathology of the lungs (Table 5)    -   3. Eradication of virus from the lungs (Table 6)    -   4. The magnitude of anti-influenza antibodies in the serum        (Table 7) and of cytokine response (FIG. 3).

TABLE 4 Effect of intranasal administration of cranberry derived NDMNumber of symptomatic mice at days post infection Experimental 0 3 7 8 914 group H/S/D H/S/D H/S/D H/S/D H/S/D H/S/D Virus 9/0/0 0/9/0 0/9/00/9/0 0/7/2 0/0/9 Virus + NDM 9/0/0 9/0/0 9/0/0 9/0/0 9/0/0 9/0/0 NDM9/0/0 9/0/0 9/0/0 9/0/0 9/0/0 9/0/0 H/S/D = Healthy/Sick/Dead

The effect of intranasal administration (inhalation) of cranberryconstituents on lungs histopathology in influenza-virus infected mice issummarized in Table 5:

TABLE 5 Lungs histopathology Bronchial Epithelial Epithelial AlveolarAlveolar Lumen sloughing necrosis necrosis infiltration Virus DebrisModerate Moderate Moderate Mild Neutrophils to marked neutrophilsVirus + Clear Rare No No Minimal to NDM no

The effect of intranasal administration (inhalation) of cranberryconstituents on virus recovery from lungs of influenza-virus infectionin mice is summarized in Table 6

TABLE 6 Virus recovery Hemagglutinating units of virus ^(a) Day 3 Day 9Day 14 Virus 6.75 2.25 Died Virus + NDM 2 0.5 0.5 ^(a) Numbers are thetiters (Log₁₀) of the Influenza virus extracted from homogenized lungsafter the indicated post infection days and determined byhemagglutination.

The effect of intranasal administration (inhalation) of cranberryconstituents on virus antibodies in serum of influenza infection mice isshown in Table 7.

TABLE 7 Virus antibodies in serum ELISA antiviral units Day 3 Day 9 Day14 Virus <10 226.3 Died Virus + NDM <10 <10 6.1

FIG. 3 shows that the magnitude of cytokine response as a result oftreatment with NMD markedly affected the infectious process induced byInfluenza virus.

Additional animal experiments showed that the above effects of cranberryconstituents were also observed when NDM was administered intranasalyafter establishing viral infection (data not shown). Unexpectedly,cranberry constituents were as potent as those of Amantadine, Amantadinebeing a commercially available antiviral medicine (data not shown).

Taken together the above results show that cranberry constituents (NDMas well as PAC) applied directly to the port of the pathogen entry intothe organ (mucosal surfaces), possess both protective and therapeuticeffects.

1. A method of treating a viral infection in a subject, comprising:administering to a virally infected subject a therapeutically effectiveamount of a pharmaceutical composition comprising a polymeric materialisolated from Vaccinium macrocarpon berry juice having a molecularweight of ≧12,000 kDa, wherein the viral infection is caused by anInfluenza virus, and wherein the polymeric material, when assayed withina preparation containing the polymeric material at a concentration of0.1 mg/ml therein, comprises less than 0.02 mg/ml of phenolic residues.2. The method of claim 1, wherein the polymeric material isolated fromVaccinium macrocarpon berry juice comprises from 43% to 51% by weightcarbon and from 4% to 5% by weight hydrogen.
 3. The method of claim 1,wherein the polymeric material isolated from Vaccinium macrocarpon berryjuice comprises: from 43% to 51% by weight carbon; from 4% to 5% byweight hydrogen; and no nitrogen, no sulfur and no chlorine.
 4. Themethod of claim 1, wherein the polymeric material isolated fromVaccinium macrocarpon berry juice exhibits an ultraviolet spectrum witha peak at 280 nm in neutral or acidic pH solutions, and wherein the peakis absent in alkali solutions.
 5. The method of claim 1, wherein thepolymeric material isolated from Vaccinium macrocarpon berry juicecomprises at least one characteristic selected from the group consistingof the polymeric material isolated from Vaccinium macrocarpon berryjuice is capable of inhibiting viral adsorption to a susceptible hostcell; the polymeric material isolated from Vaccinium macrocarpon berryjuice is capable of inhibiting viral invasion of a susceptible hostcell; and the polymeric material isolated from Vaccinium macrocarponberry juice is capable of inhibiting viral replication in a host cell.6. The method of claim 5, wherein the polymeric material isolated fromVaccinium macrocarpon berry juice comprises at least one characteristicselected from the group consisting of the polymeric material isolatedfrom Vaccinium macrocarpon berry juice is a non-dialyzable material(NDM) at a molecular weight cut-off of≧12,000 kDa-14,000 kDa; thepolymeric material isolated from Vaccinium macrocarpon berry juiceexhibits tannin-like properties; the polymeric material isolated fromVaccinium macrocarpon berry juice is highly soluble in water; thepolymeric material isolated from Vaccinium macrocarpon berry juice isfree from proteins, carbohydrates and fatty acids; and the polymericmaterial isolated from Vaccinium macrocarpon berry juice contains 56.6%by weight carbon and 4.14% by weight hydrogen atoms.
 7. The method ofclaim 5, wherein the viral adsorption to a susceptible host cell isthrough receptors comprising sialyl sugar chains present on the hostcell's surface.
 8. The method of claim 1, wherein the subject comprisesa human or an animal.
 9. The method of claim 1, wherein administeringcomprising intranasal administration.
 10. The method of claim 1, whereinthe polymeric material isolated from Vaccinium macrocarpon berry juiceand having a molecular weight≧12,000 kDa, is produced by the methodcomprising: providing a Vaccinium macrocarpon berry juice concentrate;subjecting the Vaccinium macrocarpon berry juice concentrate to dialysisat a molecular weight cut-off of ≧12,000 kDa-14,000 kDa; and collectingnon-dialyzable material at the cut-off, to obtain the polymeric materialisolated from Vaccinium macrocarpon berry juice and having a molecularweight≧12,000 kDa.